Crystalline form of a 3-phenoxymethylpyrrolidine compound

ABSTRACT

The invention provides a crystalline hydrochloride salt of (S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine. This invention also provides pharmaceutical compositions comprising the crystalline salt, processes and intermediates for preparing the crystalline salt, and methods of using the crystalline salt to treat diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/362,773, filed on Jul. 9, 2010; the entire disclosure of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel crystalline form of a3-phenoxymethylpyrrolidine compound, which has activity as a serotonin(5-HT) and norepinephrine (NE) reuptake inhibitor. This invention alsorelates to pharmaceutical compositions comprising the crystallinecompound or prepared from such compound, processes and intermediates forpreparing the crystalline compound, and methods of using such compoundto treat a pain disorder, such as neuropathic pain, and other ailments.

2. State of the Art

Pain is an unpleasant sensory and emotional experience associated withactual or potential tissue damage, or described in terms of such damage(International Association for the Study of Pain (IASP), PainTerminology). Chronic pain persists beyond acute pain or beyond theexpected time for an injury to heal (American Pain Society. “PainControl in the Primary Care Setting.” 2006:15). Neuropathic pain is paininitiated or caused by a primary lesion or dysfunction in the nervoussystem. Peripheral neuropathic pain occurs when the lesion ordysfunction affects the peripheral nervous system and centralneuropathic pain when the lesion or dysfunction affects the centralnervous system (IASP).

Several types of therapeutic agents are currently used to treatneuropathic pain including, for example, tricyclic antidepressants,serotonin and norepinephrine reuptake inhibitors, calcium channelligands (e.g., gabapentin and pregabalin), topical lidocaine, and opioidagonists (e.g., morphine, oxycodone, methadone, levorphanol andtramadol).

(S)-3-[(S)-1-(4-Chlorophenoxy)-2-methylpropyl]pyrrolidine, described incommonly-assigned U.S. patent application Ser. No. 12/834,128, filed onJul. 12, 2010 to Stangeland et al., inhibits the reuptake of bothserotonin and norepinephrine by binding to the serotonin andnorepinephrine transporters. When preparing compounds for long termstorage and when preparing pharmaceutical compositions and formulations,it is often desirable to have a crystalline form of the therapeuticagent that is neither hygroscopic nor deliquescent. It is alsoadvantageous to have a crystalline form that has a relatively highmelting point (i.e. about 128° C.), which allows the material to beprocessed, for example, micronized, without significant decomposition.Accordingly, a need exists for a stable, non-deliquescent form of(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine which has anacceptable level of hygroscopicity and a relatively high melting point.

SUMMARY OF THE INVENTION

The present invention relates to a crystalline hydrochloride salt of(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine. In oneembodiment, the invention relates to a crystalline salt of(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine andhydrochloric acid in a 1:1 molar ratio.

One aspect of the invention relates to processes for preparing acrystalline hydrochloride salt of(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine. In oneembodiment, a process for preparing a crystalline hydrochloride salt of(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine comprises thesteps of: (a) treating a hydrochloride salt of(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine with a polarsolvent to form a first composition or deprotecting(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine-1-carboxylicacid t-butyl ester with hydrochloric acid in an inert diluent to form afirst composition; and (b) adding a nonpolar solvent to form a secondcomposition from which the crystalline hydrochloride salt of theinvention is formed. In one particular embodiment, step (b) comprises:(i) adding a non-polar solvent to form a second composition; (ii)optionally cooling to effect crystallization; and (iii) isolating theresulting solids to yield the crystalline hydrochloride salt of theinvention.

Another aspect of the invention relates to a process for purifying(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine. In oneembodiment, this process comprises forming a crystalline hydrochloridesalt of (S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine. Theinvention also relates to products prepared by the processes describedherein.

One aspect of the invention relates to a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a crystallinehydrochloride salt of(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine. Suchcompositions may optionally contain other active agents such asanti-Alzheimer's agents, anticonvulsants, antidepressants,anti-Parkinson's agents, dual serotonin-norepinephrine reuptakeinhibitors, non-steroidal anti-inflammatory agents, norepinephrinereuptake inhibitors, opioid agonists, opioid antagonists, selectiveserotonin reuptake inhibitors, sodium channel blockers, sympatholytics,and combinations thereof. Accordingly, in yet another aspect of theinvention, a pharmaceutical composition comprises the crystalline saltof the invention, a second active agent, and a pharmaceuticallyacceptable carrier. Another aspect of the invention relates to acombination of active agents, comprising the crystalline salt of theinvention and a second active agent. The crystalline salt of theinvention can be formulated together or separately from the additionalagent(s). When formulated separately, a pharmaceutically acceptablecarrier may be included with the additional agent(s). Thus, yet anotheraspect of the invention relates to a combination of pharmaceuticalcompositions, the combination comprising: a first pharmaceuticalcomposition comprising the crystalline salt of the invention and a firstpharmaceutically acceptable carrier; and a second pharmaceuticalcomposition comprising a second active agent and a secondpharmaceutically acceptable carrier. The invention also relates to a kitcontaining such pharmaceutical compositions, for example where the firstand second pharmaceutical compositions are separate pharmaceuticalcompositions.

(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine possessesserotonin reuptake inhibitory activity and norepinephrine reuptakeinhibitory activity. The crystalline hydrochloride salt of this compoundis expected to have the same activity and thus the same utility as atherapeutic agent for treating patients suffering from a disease ordisorder that is treated by the inhibition of the serotonin and/or thenorepinephrine transporter. Thus, one aspect of the invention relates toa method of treating: a pain disorder such as neuropathic pain orfibromyalgia; a depressive disorder such as major depression; anaffective disorder such as an anxiety disorder; attention deficithyperactivity disorder; a cognitive disorder such as dementia; stressurinary incontinence; chronic fatigue syndrome; obesity; or vasomotorsymptoms associated with menopause, comprising administering to apatient a therapeutically effective amount of the crystalline compoundof the invention.

Yet another aspect of the invention relates to the use of thecrystalline compound of the invention for the manufacture ofmedicaments, especially for the manufacture of medicaments useful fortreating pain disorders, depressive disorders, affective disorders,attention deficit hyperactivity disorder, cognitive disorders, stressurinary incontinence, for inhibiting serotonin reuptake in a mammal, orfor inhibiting norepinephrine reuptake in a mammal. Other aspects andembodiments of the invention are disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows a powder x-ray diffraction (PXRD) pattern of thecrystalline hydrochloride salt of(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine. FIG. 2 showsa differential scanning calorimetry (DSC) thermograph. FIG. 3 shows anda thermal gravimetric analysis (TGA) trace. FIG. 4 shows a dynamicmoisture sorption (DMS) profile. FIG. 5 is a micrographic image.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a crystalline hydrochloride salt of(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine. Surprisingly,this crystalline compound has been found not to be deliquescent, evenwhen exposed to atmospheric moisture. Additionally, this crystallinecompound has an acceptable level of hygroscopicity and a high meltingpoint.

DEFINITIONS

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, and so forth used herein are tobe understood as being modified in all instances by the term “about,”unless otherwise indicated. Accordingly, the numbers set forth hereinare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each number should at least be construed in lightof the reported significant digits and by applying ordinary roundingtechniques.

As used herein, the phrase “of the formula”, “having the formula” or“having the structure” is not intended to be limiting and is used in thesame way that the term “comprising” is commonly used.

The term “melting point” as used herein means the temperature at whichthe maximum endothermic heat flow is observed by differential scanningcalorimetry, for the thermal transition that corresponds to thesolid-to-liquid phase change.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise unacceptable when used in the invention. Forexample, the term “pharmaceutically acceptable carrier” refers to amaterial that can be incorporated into a composition and administered toa patient without causing unacceptable biological effects or interactingin an unacceptable manner with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug Administration.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need thereof,i.e., the amount of drug needed to obtain the desired therapeuticeffect. For example, a therapeutically effective amount for treatingneuropathic pain is an amount of compound needed to, for example,reduce, suppress, eliminate or prevent the symptoms of neuropathic painor to treat the underlying cause of neuropathic pain. On the other hand,the term “effective amount” means an amount sufficient to obtain adesired result, which may not necessary be a therapeutic result. Forexample, when studying a system comprising a norepinephrine transporter,an “effective amount” may be the amount needed to inhibit norepinephrinereuptake.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as neuropathic pain)in a patient, such as a mammal (particularly a human), that includes oneor more of the following: (a) preventing the disease or medicalcondition from occurring, i.e., prophylactic treatment of a patient; (b)ameliorating the disease or medical condition, i.e., eliminating orcausing regression of the disease or medical condition in a patient; (c)suppressing the disease or medical condition, i.e., slowing or arrestingthe development of the disease or medical condition in a patient; or (d)alleviating the symptoms of the disease or medical condition in apatient. For example, the term “treating neuropathic pain” would includepreventing neuropathic pain from occurring, ameliorating neuropathicpain, suppressing neuropathic pain, and alleviating the symptoms ofneuropathic pain. The term “patient” is intended to include thosemammals, such as humans, that are in need of treatment or diseaseprevention, that are presently being treated for disease prevention ortreatment of a specific disease or medical condition, as well as testsubjects in which compounds of the invention are being evaluated orbeing used in a assay, for example an animal model.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

The crystalline compound of the invention can be synthesized fromreadily available starting materials as described below and in theExamples. It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. It will beappreciated that while specific process conditions (i.e. crystallizationtemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. In some instances, reactions or crystallizations wereconducted at room temperature and no actual temperature measurement wastaken. It is understood that room temperature can be taken to mean atemperature within the range commonly associated with the ambienttemperature in a laboratory environment, and will typically be in therange of about 25° C. to about 50° C. In other instances, reactions orcrystallizations were conducted at room temperature and the temperaturewas actually measured and recorded.

Generally, the crystallization is conducted in a suitable solvent. Uponcompletion of the crystallization, the crystalline compound can beisolated from the reaction mixture by any conventional means such asprecipitation, concentration, centrifugation and the like. The molarratios described in the methods of the invention can be readilydetermined by various methods available to those skilled in the art. Forexample, such molar ratios can be readily determined by ¹H NMR.Alternatively, elemental analysis and HPLC methods can be used todetermine the molar ratio.

The starting material can be readily prepared from commerciallyavailable starting materials and reagents using the procedures that arewell known in the art, and examples are provided in the Examples herein.In one embodiment, the starting material is a hydrochloride salt of(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine, which isprepared by a nucleophilic aromatic substitution reaction (S_(N)Ar),followed by deprotection with hydrochloric acid. For example, thehydrochloride salt can be prepared by dissolving(S)-3-((S)-1-hydroxy-2-methylpropyl)pyrrolidine-1-carboxylic acidt-butyl ester (1.0 eq.) and 1-chloro-4-fluorobenzene (3.0 eq.) in asuitable solvent, followed by the addition of sodium hydride (NaH, 1.5eq.). This yields a BOC-protected intermediate which can then bedeprotected with 1.20 M HCl in EtOH, to yield the desired hydrochloridesalt. In another embodiment, the starting material is(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine-1-carboxylicacid t-butyl ester, which is prepared by a Mitsunobu coupling reaction.For example, the ester can be prepared by dissolving(S)-3-((R)-1-hydroxy-2-methylpropyl)pyrrolidine-1-carboxylic acidt-butyl ester (1.0 eq.), p-chlorophenol (2.0 eq.), and a phosphinecatalyst such as triphenylphosphine (1.1 eq.) in a suitable solvent,followed by the addition of an azodicarboxylate such as diisopropylazodicarboxylate or diethyl azodicarboxylate.

In one embodiment, the crystalline hydrochloride salt of the inventioncan be prepared by a) treating a hydrochloride salt of(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine with a polarsolvent to complete dissolution and form a first composition, and b)adding a nonpolar solvent to form a second composition from which thecrystalline hydrochloride salt is formed. The polar solvent is typicallya protic solvent such as methanol, ethanol, propanol, n-propanol,isopropanol, n-butanol, ethylene glycol, water, acetic acid, formicacid, and the like. In one particular embodiment, the polar solvent isisopropanol. Generally, dissolution is conducted at an elevatedtemperature ranging from about 30-70° C., such as at a temperatureranging from about 50-60° C. In one embodiment, the solution is heatedto a temperature of about 55° C.

In another embodiment, the crystalline hydrochloride salt of theinvention can be prepared by a) deprotecting(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine-1-carboxylicacid t-butyl ester with hydrochloric acid in an inert diluent tocomplete dissolution and form a first composition, and b) adding anonpolar solvent to form a second composition from which the crystallinehydrochloride salt is formed. In one embodiment, deprotection is donewith 3 M HCl and the inert diluent is cyclopentyl methyl ether.Generally, dissolution is conducted room temperature.

Suitable nonpolar solvents for use in step (b) of the process of theinvention include, by way of illustration and not limitation, pentane,cyclopentane, hexane, cyclohexane, benzene, toluene, 1,4-dioxane,chloroform, carbon tetrachloride, diethyl ether, diisopropyl ether, anddibutyl ether, and the like. In one embodiment, the nonpolar solvent isdiisopropyl ether. The solution is then optionally cooled to form thecrystalline compound of the invention. In one particular embodiment, thesolution is cooled to about 15-30° C., and in another embodiment to atemperature of about room temperature. After a suitable amount of time,crystals will be observed. In one embodiment, crystals are observedafter a period of several hours, and in one embodiment, after a periodof about 1-3 hours. After crystals are observed, the volume of themother liquor can be reduced and the crystals isolated and dried, forexample, isolated by filtration and dried under vacuum. In oneembodiment, once crystals are observed, crystals are allowed to developfor a period of about 0.5-3 hours prior to isolation.

In another embodiment, during the cooling step, the solution is seededwith previously formed hydrochloride salt crystals. Such seed crystalscan be produced by heating the hydrochloride salt in a polar solvent,then cooling the solution in the presence of a nonpolar solvent, asdescribed above.

Crystalline Properties

Among other advantages, it has been discovered that forming acrystalline hydrochloride salt of(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine, is useful forpurifying the compound itself. For example, the crystallinehydrochloride salt of the invention has a purity of about 99%.

As is well known in the field of powder x-ray diffraction, relative peakheights of PXRD spectra are dependent on a number of factors relating tosample preparation and instrument geometry, while peak positions arerelatively insensitive to experimental details. A PXRD pattern wasobtained as set forth in Example 2. Thus, in one embodiment, thecrystalline compound of the invention is characterized by a PXRD patternhaving certain peak positions.

The crystalline compound is characterized by a PXRD pattern in which thepeak positions are substantially in accordance with those shown inFIG. 1. Those peaks are listed below, in order of descending relativeintensity. All PXRD peak intensities were corrected by subtracting thecorresponding background intensity for each peak.

I % 2-Theta 100 20.36 43 25.46 29 21.50 15 26.42 14 30.65 13 15.26 928.91 9 19.08 8 24.77 5 14.42 5 16.74 2 5.20 2 8.78

Thus, in one embodiment, the crystalline compound is characterized by apowder x-ray diffraction (PXRD) pattern comprising diffraction peaks at20 values of 8.78±0.20, 15.26±0.20, 19.08±0.20, 20.36±0.20, 21.50±0.20,and 25.46±0.20; and further characterized by having one or moreadditional diffraction peaks at 2θ values selected from 26.42±0.20,30.65±0.20, 28.91±0.20, 24.77±0.20, 14.42±0.20, 16.74±0.20, and5.20±0.20.

A differential scanning calorimetry (DSC) trace was obtained as setforth in Example 3. Thus, in one embodiment, the crystalline compound ischaracterized by its DSC thermograph. In one embodiment, the crystallinecompound is characterized by a DSC thermograph which shows a meltingpoint of about 128° C., with no significant thermal decomposition belowabout 200° C., as seen in FIG. 2.

Thermogravimetric analysis (TGA) was performed on the crystallinecompound as described in Example 3. Thus, in one embodiment, thecrystalline compound is characterized by its TGA trace. In oneembodiment, the crystalline compound is characterized by a TGA tracewhich does not show any significant amount of weight loss (which isconsistent with the loss of residual moisture or solvent) attemperatures below about 200° C., as seen in FIG. 3.

The crystalline compound of the invention has been demonstrated to havea reversible sorption/desorption profile with acceptable levels ofhygroscopicity. For example, the crystalline compound has insignificanthygroscopicity, and has exhibited less than about 1.0% weight gain whenexposed to up to 85% relative humidity, as seen in FIG. 4.

These properties of the crystalline compound of the invention arefurther illustrated in the Examples below.

UTILITY

(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine possessesserotonin and norepinephrine reuptake inhibitory activity. Thus, thiscompound, as well as of the crystalline compound of the invention, isexpected to have therapeutic utility as a combined serotonin andnorepinephrine reuptake inhibitor (SNRI).

The inhibition constant (K_(i)) of a compound is the concentration ofligand in a radioligand binding inhibition assay that would occupy 50%of the transporters if no radioligand were present. K_(i) values can bedetermined from radioligand binding studies with ³H-nisoxetine (for thenorepinephrine transporter, NET) and ³H-citalopram (for the serotonintransporter, SERT), as described in Assay 1. These K_(i) values arederived from IC₅₀ values in the binding assay using the Cheng-Prusoffequation and the IQ of the radioligand (Cheng & Prusoff (1973) Biochem.Pharmacol. 22(23):3099-3108). Functional IC₅₀ values can be determinedin the functional inhibition of uptake assays described in Assay 2.These IC₅₀ values can be converted to K_(i) values using theCheng-Prusoff equation and the K_(m) of the transmitter for thetransporter. It is noted however, that the uptake assay conditionsdescribed in Assay 2 are such that the IC₅₀ values are very close to theK_(i) values, should a mathematical conversion be desired, since theneurotransmitter concentration (5-HT, NE, or DA) used in the assay iswell below its K_(m) for the respective transporter.

Exemplary assays to determine the serotonin and/or norepinephrinereuptake inhibiting activity of compounds of the invention include byway of illustration and not limitation, assays that measure SERT and NETbinding, for example, as described in Assay 1 and in Tsuruda et al.(2010) Journal of Pharmacological and Toxicological Methods61(2):192-204. In addition, it is useful to understand the level of DATbinding and uptake in an assay such as that described in Assay 1. Usefulsecondary assays include neurotransmitter uptake assays to measureinhibition of serotonin and norepinephrine uptake into cells expressingthe respective human or rat recombinant transporter (hSERT, hNET, orhDAT) as described in Assay 2, and ex vivo radioligand binding andneurotransmitter uptake assays that are used to determine the in vivooccupancy of SERT, NET and DAT in tissue as described in Assay 3. Otherassays that are useful to evaluate pharmacological properties of testcompounds include those listed in Assay 4. Exemplary in vivo assaysinclude the formalin paw test described in Assay 5, which is a reliablepredictor of clinical efficacy for the treatment of neuropathic pain,and the spinal nerve ligation model described in Assay 6. Theaforementioned assays are useful in determining the therapeutic utility,for example, the neuropathic pain relieving activity, of compounds ofthe invention. Other properties and utilities of compounds of theinvention can be demonstrated using various in vitro and in vivo assayswell-known to those skilled in the art.

The crystalline compound of the invention is expected to be useful forthe treatment and/or prevention of medical conditions in which theregulation of monoamine transporter function is implicated, inparticular those conditions mediated by or responsive to the inhibitionof serotonin and norepinephrine reuptake. Thus it is expected thatpatients suffering from a disease or disorder that is treated by theinhibition of the serotonin and/or the norepinephrine transporter can betreated by administering a therapeutically effective amount of thecrystalline compound of the invention. Such medical conditions include,by way of example, pain disorders such as neuropathic pain,fibromyalgia, and chronic pain, depressive disorders such as majordepression, affective disorders such as an anxiety disorder, attentiondeficit hyperactivity disorder, cognitive disorders such as dementia,and stress urinary incontinence.

The amount of active agent administered per dose or the total amountadministered per day may be predetermined or it may be determined on anindividual patient basis by taking into consideration numerous factors,including the nature and severity of the patient's condition, thecondition being treated, the age, weight, and general health of thepatient, the tolerance of the patient to the active agent, the route ofadministration, pharmacological considerations such as the activity,efficacy, pharmacokinetics and toxicology profiles of the active agentand any secondary agents being administered, and the like. Treatment ofa patient suffering from a disease or medical condition (such asneuropathic pain) can begin with a predetermined dosage or a dosagedetermined by the treating physician, and will continue for a period oftime necessary to prevent, ameliorate, suppress, or alleviate thesymptoms of the disease or medical condition. Patients undergoing suchtreatment will typically be monitored on a routine basis to determinethe effectiveness of therapy. For example, in treating neuropathic pain,a measure of the effectiveness of treatment may involve assessment ofthe patient's quality of life, e.g., improvements in the patient'ssleeping patterns, work attendance, ability to exercise and beambulatory, etc. Pain scales, operating on a point basis, may also beused to help evaluate a patient's pain level. Indicators for the otherdiseases and conditions described herein, are well-known to thoseskilled in the art, and are readily available to the treating physician.Continuous monitoring by the physician will insure that the optimalamount of active agent will be administered at any given time, as wellas facilitating the determination of the duration of treatment. This isof particular value when secondary agents are also being administered,as their selection, dosage, and duration of therapy may also requireadjustment. In this way, the treatment regimen and dosing schedule canbe adjusted over the course of therapy so that the lowest amount ofactive agent that exhibits the desired effectiveness is administeredand, further, that administration is continued only so long as isnecessary to successfully treat the disease or medical condition.

Pain Disorders

SNRIs have been shown to have a beneficial effect on pain such aspainful diabetic neuropathy (duloxetine, Goldstein et al. (2005) Pain116:109-118; venlafaxine, Rowbotham et al. (2004) Pain 110:697-706),fibromyalgia (duloxetine, Russell et al. (2008) Pain 136(3):432-444;milnacipran, Vitton et al. (2004) Human Psychopharmacology 19:S27-S35),and migraine (venlafaxine, Ozyalcin et al. (2005) Headache45(2):144-152). Thus, one embodiment of the invention relates to amethod for treating a pain disorder, comprising administering to apatient a therapeutically effective amount of the crystalline compoundof the invention. Typically, the therapeutically effective amount willbe the amount that is sufficient to relieve the pain. Exemplary paindisorders include, by way of illustration, acute pain, persistent pain,chronic pain, inflammatory pain, and neuropathic pain. Morespecifically, these include pain associated with or caused by:arthritis; back pain including chronic low back pain; cancer, includingtumor related pain (e.g., bone pain, headache, facial pain or visceralpain) and pain associated with cancer therapy (e.g., post-chemotherapysyndrome, chronic post-surgical pain syndrome and post-radiationsyndrome); carpal tunnel syndrome; fibromyalgia; headaches includingchronic tension headaches; inflammation associated with polymyalgia,rheumatoid arthritis and osteoarthritis; migraine; neuropathic painincluding complex regional pain syndrome; overall pain; post-operativepain; shoulder pain; central pain syndromes, including post-stroke pain,and pain associated with spinal cord injuries and multiple sclerosis;phantom limb pain; pain associated with Parkinson's disease; andvisceral pain (e.g., irritable bowel syndrome). Of particular interestis the treatment of neuropathic pain, which includes diabetic peripheralneuropathy (DPN), HIV-related neuropathy, post-herpetic neuralgia (PHN),and chemotherapy-induced peripheral neuropathy. When used to treat paindisorders such as neuropathic pain, compounds of the invention may beadministered in combination with other therapeutic agents, includinganticonvulsants, antidepressants, muscle relaxants, NSAIDs, opioidagonists, opioid antagonists, selective serotonin reuptake inhibitors,sodium channel blockers, and sympatholytics. Exemplary compounds withinthese classes are described herein.

Depressive Disorders

Another embodiment of the invention relates to a method of treating adepressive disorder, comprising administering to a patient atherapeutically effective amount of the crystalline compound of theinvention. Typically, the therapeutically effective amount will be theamount that is sufficient to alleviate depression and provide a sense ofgeneral well-being. Exemplary depressive disorders include, by way ofillustration and not limitation: depression associated with Alzheimer'sdisease, bipolar disorder, cancer, child abuse, infertility, Parkinson'sdisease, postmyocardial infarction, and psychosis; dysthymia; grumpy orirritable old man syndrome; induced depression; major depression;pediatric depression; postmenopausal depression; post partum depression;recurrent depression; single episode depression; and subsyndromalsymptomatic depression. Of particular interest is the treatment of majordepression. When used to treat depressive disorders, compounds of theinvention may be administered in combination with other therapeuticagents, including antidepressants and dual serotonin-norepinephrinereuptake inhibitors. Exemplary compounds within these classes aredescribed herein.

Affective Disorders

Another embodiment of the invention relates to a method of treating anaffective disorder, comprising administering to a patient atherapeutically effective amount of the crystalline compound of theinvention. Exemplary affective disorders include, by way of illustrationand not limitation: anxiety disorders such as general anxiety disorder;avoidant personality disorder; eating disorders such as anorexianervosa, bulimia nervosa and obesity; obsessive compulsive disorder;panic disorder; personality disorders such as avoidant personalitydisorder and attention deficit hyperactivity disorder (ADHD);post-traumatic stress syndrome; phobias such as agoraphobia, as well assimple and other specific phobias, and social phobia; premenstrualsyndrome; psychotic disorders, such as schizophrenia and mania; seasonalaffective disorder; sexual dysfunction, including premature ejaculation,male impotence, and female sexual dysfunction such as female sexualarousal disorder; social anxiety disorder; and substance abusedisorders, including chemical dependencies such as addictions toalcohol, benzodiazepines, cocaine, heroin, nicotine and phenobarbital,as well as withdrawal syndromes that may arise from these dependencies.When used to treat affective disorders, compounds of the invention maybe administered in combination with other therapeutic agents, includingantidepressants. Exemplary compounds within these classes are describedherein.

Atomoxetine, which is 10-fold NET selective, is approved for attentiondeficit hyperactivity disorder (ADHD) therapy, and clinical studies haveshown that the SNRI, venlafaxine, can also have a beneficial effect intreating ADHD (Mukaddes et al. (2002) Eur. Neuropsychopharm. 12(Supp3):421). Thus, the crystalline compound of the invention is alsoexpected to be useful in methods for treating attention deficithyperactivity disorder by administering to a patient a therapeuticallyeffective amount of the crystalline compound of the invention. When usedto treat depression, the crystalline compound of the invention may beadministered in combination with other therapeutic agents, includingantidepressants. Exemplary compounds within these classes are describedherein.

Cognitive Disorders

Another embodiment of the invention relates to a method of treating acognitive disorder, comprising administering to a patient atherapeutically effective amount of the crystalline compound of theinvention. Exemplary cognitive disorders include, by way of illustrationand not limitation: dementia, which includes degenerative dementia(e.g., Alzheimer's disease, Creutzfeldt-Jakob disease, Huntingdon'schorea, Parkinson's disease, Pick's disease, and senile dementia),vascular dementia (e.g., multi-infarct dementia), and dementiaassociated with intracranial space occupying lesions, trauma, infectionsand related conditions (including HIV infection), metabolism, toxins,anoxia and vitamin deficiency; and mild cognitive impairment associatedwith ageing, such as age associated memory impairment, amnesiac disorderand age-related cognitive decline. When used to treat cognitivedisorders, the crystalline compound of the invention may be administeredin combination with other therapeutic agents, including anti-Alzheimer'sagents and anti-Parkinson's agents. Exemplary compounds within theseclasses are described herein.

Other Disorders

SNRIs have also been shown to be effective for the treatment of stressurinary incontinence (Dmochowski (2003) Journal of Urology 170(4):1259-1263). Thus, another embodiment of the invention relates to amethod for treating stress urinary incontinence, comprisingadministering to a patient a therapeutically effective amount of thecrystalline compound of the invention. When used to treat stress urinaryincontinence, compounds of the invention may be administered incombination with other therapeutic agents, including anticonvulsants.Exemplary compounds within these classes are described herein.

Duloxetine, an SNRI, is undergoing clinical trials for evaluating itsefficacy in treating chronic fatigue syndrome, and has recently beenshown to be effective in treating fibromyalgia (Russell et al. (2008)Pain 136(3):432-444). The crystalline compound of the invention, due toits expected ability to inhibit SERT and NET, is also expected to havethis utility, and another embodiment of the invention relates to amethod for treating chronic fatigue syndrome, comprising administeringto a patient a therapeutically effective amount of the crystallinecompound of the invention.

Sibutramine, a norepinephrine and dopamine reuptake inhibitor, has beenshown to be useful in treating obesity (Wirth et al. (2001) JAMA286(11):1331-1339). The crystalline compound of the invention, due toits expected ability to inhibit NET, is also expected to have thisutility, and another embodiment of the invention relates to a method fortreating obesity, comprising administering to a patient atherapeutically effective amount of the crystalline compound of theinvention.

Desvenlafaxine, an SNRI, has been shown to relieve vasomotor symptomsassociated with menopause (Deecher et al. (2007) Endocrinology148(3):1376-1383). The crystalline compound of the invention, due to itsexpected ability to inhibit SERT and NET, is also expected to have thisutility, and another embodiment of the invention relates to a method fortreating vasomotor symptoms associated with menopause, comprisingadministering to a patient a therapeutically effective amount of thecrystalline compound of the invention.

Research Tools

Since the crystalline compound of the invention is expected to possessboth serotonin reuptake inhibition activity and norepinephrine reuptakeinhibition activity, this compound is also expected to find utility as aresearch tool for investigating or studying biological systems orsamples having serotonin or norepinephrine transporters. Any suitablebiological system or sample having serotonin and/or norepinephrinetransporters may be employed in such studies which may be conductedeither in vitro or in vivo. Representative biological systems or samplessuitable for such studies include, but are not limited to, cells,cellular extracts, plasma membranes, tissue samples, isolated organs,mammals (such as mice, rats, guinea pigs, rabbits, dogs, pigs, humans,and so forth), and the like, with mammals being of particular interest.In one particular embodiment of the invention, serotonin reuptake in amammal is inhibited by administering a serotonin reuptake-inhibitingamount of the crystalline compound of the invention. In anotherparticular embodiment, norepinephrine reuptake in a mammal is inhibitedby administering a norepinephrine reuptake-inhibiting amount of thecrystalline compound of the invention. The crystalline compound of theinvention can also be used as a research tool by conducting biologicalassays using such compound.

When used as a research tool, a biological system or sample comprising aserotonin transporter and/or a norepinephrine transporter is typicallycontacted with a serotonin reuptake-inhibiting or norepinephrinereuptake-inhibiting amount of the crystalline compound of the invention.After the biological system or sample is exposed to the compound, theeffects of inhibiting serotonin reuptake and/or norepinephrine reuptakeare determined using conventional procedures and equipment. Exposureencompasses contacting cells or tissue with the compound, administeringthe compound to a mammal, for example by i.p. or i.v. administration,and so forth. This determining step may comprise measuring a response,i.e., a quantitative analysis or may comprise an observation, i.e., aqualitative analysis. Measuring a response involves, for example,determining the effects of the compound on the biological system orsample using conventional procedures and equipment, such as serotoninand norepinephrine reuptake assays. The assay results can be used todetermine the activity level as well as the amount of compound necessaryto achieve the desired result, i.e., a serotonin reuptake-inhibiting anda norepinephrine reuptake-inhibiting amount.

Additionally, the crystalline compound of the invention can be used as aresearch tool for evaluating other chemical compounds, and thus is alsouseful in screening assays to discover, for example, new compoundshaving both serotonin reuptake-inhibiting activity and norepinephrinereuptake-inhibiting activity. In this manner, the crystalline compoundof the invention is used as a standard in an assay to allow comparisonof the results obtained with a test compound and with the crystallinecompound of the invention to identify those test compounds that haveabout equal or superior reuptake-inhibiting activity, if any. Forexample, reuptake data for a test compound or a group of test compoundsis compared to the reuptake data for the crystalline compound of theinvention to identify those test compounds that have the desiredproperties, e.g., test compounds having reuptake-inhibiting activityabout equal or superior to the crystalline compound of the invention, ifany. This aspect of the invention includes, as separate embodiments,both the generation of comparison data (using the appropriate assays)and the analysis of the test data to identify test compounds ofinterest. Thus, a test compound can be evaluated in a biological assay,by a method comprising the steps of: (a) conducting a biological assaywith a test compound to provide a first assay value; (b) conducting thebiological assay with the crystalline compound of the invention toprovide a second assay value; wherein step (a) is conducted eitherbefore, after or concurrently with step (b); and (c) comparing the firstassay value from step (a) with the second assay value from step (b).Exemplary biological assays include serotonin and norepinephrinereuptake assays.

Pharmaceutical Compositions and Formulations

The crystalline compound of the invention is typically administered to apatient in the form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to, oral,rectal, vaginal, nasal, inhaled, topical (including transdermal) andparenteral modes of administration. However, it will be understood bythose skilled in the art that, once the crystalline compound of theinvention has been formulated, it may no longer be in crystalline form,i.e., it may be dissolved in a suitable carrier. Further, thecrystalline compound of the invention may be administered, for exampleorally, in multiple doses per day (e.g., twice, three times or fourtimes daily), in a single daily dose, in a twice daily dose, in a singleweekly dose, and so forth.

Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and the crystalline compound of the invention. The compositionsmay contain other therapeutic and/or formulating agents if desired. Whendiscussing compositions, the “crystalline compound of the invention” mayalso be referred to herein as the “active agent,” to distinguish it fromother components of the formulation, such as the carrier.

Pharmaceutical compositions of the invention typically contain atherapeutically effective amount of the crystalline compound of theinvention. Those skilled in the art will recognize, however, that apharmaceutical composition may contain more than a therapeuticallyeffective amount, i.e., bulk compositions, or less than atherapeutically effective amount, i.e., individual unit doses designedfor multiple administration to achieve a therapeutically effectiveamount. Typically, the composition will contain from about 0.01-95 wt %of active agent, including, from about 0.01-30 wt %, such as from about0.01-10 wt %, with the actual amount depending upon the formulationitself, the route of administration, the frequency of dosing, and soforth. In one embodiment, a composition suitable for an oral dosageform, for example, may contain about 5-70 wt %, or from about 10-60 wt %of active agent. In one exemplary embodiment, a pharmaceuticalcomposition contains from about 1 to 20 mg of active agent, includingfrom about 1 to 15 mg of active agent and from about 1 to 10 mg ofactive agent. In another exemplary embodiment, a pharmaceuticalcomposition contains from about 5 to 20 mg of active agent, includingfrom about 7.5 to 15 mg of active agent. For example the active agentmay be formulated in 1 mg and 10 mg unit doses.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers, and the like, usingconventional procedures and equipment.

In one embodiment, the pharmaceutical compositions are suitable for oraladministration. One exemplary dosing regimen would be an oral dosageform administered once or twice daily. Suitable compositions for oraladministration may be in the form of capsules, tablets, pills, lozenges,cachets, dragees, powders, granules; solutions or suspensions in anaqueous or non-aqueous liquid; oil-in-water or water-in-oil liquidemulsions; elixirs or syrups; and the like; each containing apredetermined amount of the active agent.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills, and the like), the composition will typicallycomprise the active agent and one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate. Solid dosageforms may also comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; solution retarding agents, such as paraffin;absorption accelerators, such as quaternary ammonium compounds; wettingagents, such as cetyl alcohol and/or glycerol monostearate; absorbents,such as kaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodiumsulfite, and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, if appropriate, with oneor more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Suspensions maycontain suspending agents such as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, i.e., each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

In another embodiment, the compositions of the invention are suitablefor inhaled administration, and will typically be in the form of anaerosol or a powder. Such compositions are generally administered usingwell-known delivery devices, such as a nebulizer, dry powder, ormetered-dose inhaler. Nebulizer devices produce a stream of highvelocity air that causes the composition to spray as a mist that iscarried into a patient's respiratory tract. An exemplary nebulizerformulation comprises the active agent dissolved in a carrier to form asolution, or micronized and combined with a carrier to form a suspensionof micronized particles of respirable size. Dry powder inhalersadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. An exemplary dry powderformulation comprises the active agent dry-blended with an excipientsuch as lactose, starch, mannitol, dextrose, polylactic acid,polylactide-co-glycolide, and combinations thereof. Metered-doseinhalers discharge a measured amount of the active agent usingcompressed propellant gas. An exemplary metered-dose formulationcomprises a solution or suspension of the active agent in a liquefiedpropellant, such as a chlorofluorocarbon or hydrofluoroalkane. Optionalcomponents of such formulations include co-solvents, such as ethanol orpentane, and surfactants, such as sorbitan trioleate, oleic acid,lecithin, and glycerin. Such compositions are typically prepared byadding chilled or pressurized hydrofluoroalkane to a suitable containercontaining the active agent, ethanol (if present) and the surfactant (ifpresent). To prepare a suspension, the active agent is micronized andthen combined with the propellant. Alternatively, a suspensionformulation can be prepared by spray drying a coating of surfactant onmicronized particles of the active agent. The formulation is then loadedinto an aerosol canister, which forms a portion of the inhaler.

The crystalline compound of the invention can also be administeredparenterally (e.g., by subcutaneous, intravenous, intramuscular, orintraperitoneal injection). For such administration, the active agent isprovided in a sterile solution, suspension, or emulsion. Exemplarysolvents for preparing such formulations include water, saline, lowmolecular weight alcohols such as propylene glycol, polyethylene glycol,oils, gelatin, fatty acid esters such as ethyl oleate, and the like. Atypical parenteral formulation is a sterile pH 4-7 aqueous solution ofthe active agent. Parenteral formulations may also contain one or moresolubilizers, stabilizers, preservatives, wetting agents, emulsifiers,and dispersing agents. These formulations may be rendered sterile by useof a sterile injectable medium, a sterilizing agent, filtration,irradiation, or heat.

The crystalline compound of the invention can also be administeredtransdermally using known transdermal delivery systems and excipients.For example, the compound can be admixed with permeation enhancers, suchas propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones, and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

If desired, the crystalline compound of the invention may beadministered in combination with one or more other therapeutic agents.Thus, in one embodiment, compositions of the invention may optionallycontain other drugs that are co-administered with the crystallinecompound of the invention. For example, the composition may furthercomprise one or more drugs (also referred to as “secondary agents(s)”)selected from the group of anti-Alzheimer's agents, anticonvulsants(antiepileptics), antidepressants, anti-Parkinson's agents, dualserotonin-norepinephrine reuptake inhibitors (SNRIs), non-steroidalanti-inflammatory agents (NSAIDs), norepinephrine reuptake inhibitors,opioid agonists (opioid analgesics), opioid antagonists, selectiveserotonin reuptake inhibitors, sodium channel blockers, sympatholytics,and combinations thereof. Numerous examples of such therapeutic agentsare well known in the art, and examples are described herein. Bycombining the crystalline compound of the invention with a secondaryagent, triple therapy can be achieved, i.e., serotonin reuptakeinhibitory activity, norepinephrine reuptake inhibitory activity, andactivity associated with the secondary agent (e.g., antidepressantactivity), using only two active components. Since pharmaceuticalcompositions containing two active components are typically easier toformulate than compositions containing three active components, suchtwo-component compositions provide a significant advantage overcompositions containing three active components. Accordingly, in yetanother aspect of the invention, a pharmaceutical composition comprisesthe crystalline compound of the invention, a second active agent, and apharmaceutically acceptable carrier. Third, fourth, etc., active agentsmay also be included in the composition. In combination therapy, theamount of compound of the invention that is administered, as well as theamount of secondary agents, may be less than the amount typicallyadministered in monotherapy.

The crystalline compound of the invention may be either physically mixedwith the second active agent to form a composition containing bothagents; or each agent may be present in separate and distinctcompositions which are administered to the patient simultaneously orsequentially. For example, the crystalline compound of the invention canbe combined with a second active agent using conventional procedures andequipment to form a combination of active agents comprising thecrystalline compound of the invention and a second active agent.Additionally, the active agents may be combined with a pharmaceuticallyacceptable carrier to form a pharmaceutical composition comprising thecrystalline compound of the invention, a second active agent and apharmaceutically acceptable carrier. In this embodiment, the componentsof the composition are typically mixed or blended to create a physicalmixture. The physical mixture is then administered in a therapeuticallyeffective amount using any of the routes described herein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention, ranging anywhere from concurrent withadministration of the compound of the invention to about 24 hourspost-dose. This is also referred to as sequential administration. Thus,the crystalline compound of the invention can be orally administeredsimultaneously or sequentially with another active agent using twotablets, with one tablet for each active agent, where sequential maymean being administered immediately after administration of the compoundof the invention or at some predetermined time later (e.g., one hourlater or three hours later). Alternatively, the combination may beadministered by different routes of administration, i.e., one orally andthe other by inhalation.

In one embodiment, the kit comprises a first dosage form comprising thecrystalline compound of the invention and at least one additional dosageform comprising one or more of the secondary agents set forth herein, inquantities sufficient to carry out the methods of the invention. Thefirst dosage form and the second (or third, etc.) dosage form togethercomprise a therapeutically effective amount of active agents for thetreatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount, i.e., are typically administered in an amount thatproduces a therapeutically beneficial effect when co-administered withthe crystalline compound of the invention. The secondary agent can be inthe form of a pharmaceutically acceptable salt, solvate, optically purestereoisomer, and so forth. Thus, secondary agents listed below areintended to include all such forms, and are commercially available orcan be prepared using conventional procedures and reagents.

Representative anti-Alzheimer's agents include, but are not limited to:donepezil, galantamine, memantine, rivastigmine, selegiline, tacrine,and combinations thereof.

Representative anticonvulsants (antiepileptics) include, but are notlimited to: acetazolamide, albutoin, 4-amino-3-hydroxybutyric acid,beclamide, carbamazepine, cinromide, clomethiazole, clonazepam,diazepam, dimethadione, eterobarb, ethadione, ethosuximide, ethotoin,felbamate, fosphenyloin, gabapentin, lacosamide, lamotrigine, lorazepam,magnesium bromide, magnesium sulfate, mephenyloin, mephobarbital,methsuximide, midazolam, nitrazepam, oxazepam, oxcarbazepine,paramethadione, phenacemide, pheneturide, phenobarbital, phensuximide,phenyloin, potassium bromide, pregabalin, primidone, progabide, sodiumbromide, sodium valproate, sulthiame, tiagabine, topiramate,trimethadione, valproic acid, valpromide, vigabatrin, zonisamide, andcombinations thereof. In a particular embodiment, the anticonvulsant isselected from carbamazepine, gabapentin, pregabalin, and combinationsthereof.

Representative antidepressants include, but are not limited to:adinazolam, amitriptyline, clomipramine, desipramine, dothiepin (e.g.,dothiepin hydrochloride), doxepin, imipramine, lofepramine, mirtazapine,nortriptyline, protriptyline, trimipramine, venlafaxine, zimelidine, andcombinations thereof.

Representative anti-Parkinson's agents include, but are not limited to:amantadine, apomorphine, benztropine, bromocriptine, carbidopa,diphenhydramine, entacapone, levodopa, pergolide, pramipexole,ropinirole, selegiline, tolcapone, trihexyphenidyl, and combinationsthereof.

Representative dual serotonin-norepinephrine reuptake inhibitors (SNRIs)include, but are not limited to: bicifadine, desvenlafaxine, duloxetine,milnacipran, nefazodone, venlafaxine, and combinations thereof.

Representative non-steroidal anti-inflammatory agents (NSAIDs) include,but are not limited to: acemetacin, acetaminophen, acetyl salicylicacid, alclofenac, alminoprofen, amfenac, amiprilose, amoxiprin,anirolac, apazone, azapropazone, benorilate, benoxaprofen, bezpiperylon,broperamole, bucloxic acid, carprofen, clidanac, diclofenac, diflunisal,diftalone, enolicam, etodolac, etoricoxib, fenbufen, fenclofenac,fenclozic acid, fenoprofen, fentiazac, feprazone, flufenamic acid,flufenisal, fluprofen, flurbiprofen, furofenac, ibufenac, ibuprofen,indomethacin, indoprofen, isoxepac, isoxicam, ketoprofen, ketorolac,lofemizole, lornoxicam, meclofenamate, meclofenamic acid, mefenamicacid, meloxicam, mesalamine, miroprofen, mofebutazone, nabumetone,naproxen, niflumic acid, nimesulide, nitroflurbiprofen, olsalazine,oxaprozin, oxpinac, oxyphenbutazone, phenylbutazone, piroxicam,pirprofen, pranoprofen, salsalate, sudoxicam, sulfasalazine, sulindac,suprofen, tenoxicam, tiopinac, tiaprofenic acid, tioxaprofen, tolfenamicacid, tolmetin, triflumidate, zidometacin, zomepirac, and combinationsthereof. In a particular embodiment, the NSAID is selected frometodolac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac,meloxicam, naproxen, oxaprozin, piroxicam, and combinations thereof. Ina particular embodiment, the NSAID is selected from ibuprofen,indomethacin, nabumetone, naproxen (for example, naproxen sodium), andcombinations thereof.

Representative muscle relaxants include, but are not limited to:carisoprodol, chlorzoxazone, cyclobenzaprine, diflunisal, metaxalone,methocarbamol, and combinations thereof.

Representative norepinephrine reuptake inhibitors include, but are notlimited to: atomoxetine, buproprion and the buproprion metabolitehydroxybuproprion, maprotiline, reboxetine (for example,(S,S)-reboxetine), viloxazine, and combinations thereof. In a particularembodiment, the norepinephrine reuptake inhibitor is selected fromatomoxetine, reboxetine, and combinations thereof.

Representative opioid agonists (opioid analgesics) and antagonistsinclude, but are not limited to: buprenorphine, butorphanol, codeine,dihydrocodeine, fentanyl, hydrocodone, hydromorphone, levallorphan,levorphanol, meperidine, methadone, morphine, nalbuphine, nalmefene,nalorphine, naloxone, naltrexone, nalorphine, oxycodone, oxymorphone,pentazocine, propoxyphene, tramadol, and combinations thereof. Incertain embodiments, the opioid agonist is selected from codeine,dihydrocodeine, hydrocodone, hydromorphone, morphine, oxycodone,oxymorphone, tramadol, and combinations thereof.

Representative selective serotonin reuptake inhibitors (SSRIs) include,but are not limited to: citalopram and the citalopram metabolitedesmethylcitalopram, dapoxetine, escitalopram (e.g., escitalopramoxalate), fluoxetine and the fluoxetine desmethyl metabolitenorfluoxetine, fluvoxamine (e.g., fluvoxamine maleate), paroxetine,sertraline and the sertraline metabolite demethylsertraline, andcombinations thereof. In certain embodiments, the SSRI is selected fromcitalopram, paroxetine, sertraline, and combinations thereof.

Representative sodium channel blockers include, but are not limited to:carbamazepine, fosphenyloin, lamotrignine, lidocaine, mexiletine,oxcarbazepine, phenyloin, and combinations thereof.

Representative sympatholytics include, but are not limited to: atenolol,clonidine, doxazosin, guanethidine, guanfacine, modafinil, phentolamine,prazosin, reserpine, tolazoline (e.g., tolazoline hydrochloride),tamsulosin, and combinations thereof.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

Exemplary Hard Gelatin Capsules for Oral Administration

The crystalline compound of the invention (50 g), spray-dried lactose(440 g) and magnesium stearate (10 g) are thoroughly blended. Theresulting composition is then loaded into hard gelatin capsules (500 mgof composition per capsule).

Alternately, the crystalline compound (20 mg) is thoroughly blended withstarch (89 mg), microcrystalline cellulose (89 mg) and magnesiumstearate (2 mg). The mixture is then passed through a No. 45 mesh U.S.sieve and loaded into a hard gelatin capsule (200 mg of composition percapsule).

Exemplary Gelatin Capsule Formulation for Oral Administration

The crystalline compound of the invention (100 mg) is thoroughly blendedwith polyoxyethylene sorbitan monooleate (50 mg) and starch powder (250mg). The mixture is then loaded into a gelatin capsule (400 mg ofcomposition per capsule).

Alternately, the crystalline compound (40 mg) is thoroughly blended withmicrocrystalline cellulose (Avicel PH 103; 259.2 mg) and magnesiumstearate (0.8 mg). The mixture is then loaded into a gelatin capsule(Size #1, White, Opaque) (300 mg of composition per capsule).

Exemplary Tablet Formulation for Oral Administration

The crystalline compound of the invention (10 mg), starch (45 mg) andmicrocrystalline cellulose (35 mg) are passed through a No. 20 mesh U.S.sieve and mixed thoroughly. The granules so produced are dried at 50-60°C. and passed through a No. 16 mesh U.S. sieve. A solution ofpolyvinylpyrrolidone (4 mg as a 10% solution in sterile water) is mixedwith sodium carboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg),and talc (1 mg), and this mixture is then passed through a No. 16 meshU.S. sieve. The sodium carboxymethyl starch, magnesium stearate and talcare then added to the granules. After mixing, the mixture is compressedon a tablet machine to afford a tablet weighing 100 mg.

Alternately, the crystalline compound (250 mg) is thoroughly blendedwith microcrystalline cellulose (400 mg), silicon dioxide fumed (10 mg),and stearic acid (5 mg). The mixture is then compressed to form tablets(665 mg of composition per tablet).

Alternately, the crystalline compound (400 mg) is thoroughly blendedwith cornstarch (50 mg), croscarmellose sodium (25 mg), lactose (120mg), and magnesium stearate (5 mg). The mixture is then compressed toform a single-scored tablet (600 mg of compositions per tablet).

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of active agent per 10 mL of suspension:

Ingredients Amount Crystalline compound of the invention 1.0 g Fumaricacid 0.5 g Sodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben0.05 g Granulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum ®K (magnesium 1.0 g aluminum silicate) Flavoring 0.035 mL Colorings 0.5mg Distilled water q.s. to 100 mL

Exemplary Injectable Formulation for Administration by Injection

The crystalline compound of the invention (0.2 g) is blended with 0.4 Msodium acetate buffer solution (2.0 mL). The pH of the resultingsolution is adjusted to pH 4 using 0.5 N aqueous hydrochloric acid or0.5 N aqueous sodium hydroxide, as necessary, and then sufficient waterfor injection is added to provide a total volume of 20 mL. The mixtureis then filtered through a sterile filter (0.22 micron) to provide asterile solution suitable for administration by injection.

Exemplary Compositions for Administration by Inhalation

The crystalline compound of the invention (0.2 mg) is micronized andthen blended with lactose (25 mg). This blended mixture is then loadedinto a gelatin inhalation cartridge. The contents of the cartridge areadministered using a dry powder inhaler, for example.

Alternately, a micronized compound of the invention (10 g) is dispersedin a solution prepared by dissolving lecithin (0.2 g) in demineralizedwater (200 mL). The resulting suspension is spray dried and thenmicronized to form a micronized composition comprising particles havinga mean diameter less than about 1.5 μm. The micronized composition isthen loaded into metered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μgto about 500 μg of the compound of the invention per dose whenadministered by the inhaler.

Alternately, the crystalline compound (25 mg) is dissolved in citratebuffered (pH 5) isotonic saline (125 mL). The mixture is stirred andsonicated until the compound is dissolved. The pH of the solution ischecked and adjusted, if necessary, to pH 5 by slowly adding aqueous 1Nsodium hydroxide. The solution is administered using a nebulizer devicethat provides about 10 μg to about 500 μg of the crystalline compoundper dose.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard meaning:

-   -   BOC t-butoxycarbonyl    -   BSA bovine serum albumin    -   DMEM Dulbecco's Modified Eagle's Medium    -   DMF N,N-dimethylformamide    -   DMSO dimethylsulfoxide    -   EDTA ethylenediaminetetraacetic acid    -   EtOAc ethyl acetate    -   EtOH ethanol    -   FBS fetal bovine serum    -   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   PBS phosphate buffered saline    -   THF tetrahydrofuran

Any other abbreviations used herein but not defined have their standard,generally accepted meaning. Unless noted otherwise, all materials, suchas reagents, starting materials and solvents, were purchased fromcommercial suppliers (such as Sigma-Aldrich, Fluka Riedel-de Haën, andthe like) and were used without further purification.

In the compounds described below, the two chiral centers are identifiedby the * and ** symbols. Note that the * chiral center is known;however, the ** chiral center is not known unambiguously and is basedupon the first elution peak by reverse phase HPLC from the mixture ofdiastereomeric intermediates (the protected alcohols).

Preparation 1(S)-3-(S)-1-Hydroxy-2-methylpropyl)pyrrolidine-1-carboxylic Acid t-ButylEster

(S)-3-Formylpyrrolidine-1-carboxylic acid t-butyl ester (10.0 g, 50.2mmol) and THF (100 mL, 1000 mmol) were combined under nitrogen, and theresulting solution was cooled to −78° C. 2.0M isopropylmagnesiumchloride in THF (30.1 mL, 60.2 mmol) was then added dropwise over 10minutes. The mixture was allowed to warm to room temperature slowlyovernight. Then saturated aqueous NH₄Cl (100 mL) was added dropwise toquench the reaction. The THF was removed under vacuum and the resultingmixture was extracted with EtOAc (2×100 mL), and the combined organiclayers were washed with saturated aqueous NaCl (1×100 mL), then driedover anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The crudeproduct was purified by normal phase chromatography (300 g SiO₂, 12 gcrude, 50-60% diethyl ether in hexanes) to yield the following as clearoils:

(S)-3-((S)-1-hydroxy-2-methylpropyl)pyrrolidine-1-carboxylic acidt-butyl ester (3.8 g; 1^(st) eluting peak). ¹H NMR (400 MHz, DMSO) δ4.60 4.38 (brs, 1H), 3.40-3.22 (m, 2H), 3.28-3.02 (m, 2H), 2.94-2.82 (m,1H), 2.28-2.12 (m, 1H), 1.92-1.82 (m, 1H), 1.70-1.56 (m, 1H), 1.52-1.44(m, 1H), 1.38 (s, 9H), 0.87 (d, J=6.8 Hz, 3H), 0.83 (d, J=6.8 Hz, 3H).

(S)-3-(R)-1-hydroxy-2-methylpropyl)pyrrolidine-1-carboxylic acid t-butylester (2.8 g; 2^(nd) eluting peak). ¹H NMR (400 MHz, DMSO) δ 4.50-4.40,(brs, 1H) 3.42-3.28 (m, 2H), 3.18-3.06 (m, 2H), 3.04-2.92 (m, 1H),2.26-2.12 (m, 1H), 1.78-1.68 (m, 1H), 1.62-1.46 (m, 2H), 1.38 (s, 9H),0.88 (d, J=6.8 Hz, 3H), 0.82 (d, J=6.7 Hz, 3H).

Assignment of the stereochemistry of the title compound was done by theMosher ester analysis (Dale and Mosher (1969) J. Org. Chem.34(9):2543-2549) on the material that eluted second. Using thisanalysis, the 2^(nd) eluting peak material was determined to be (S,R):

δ (S, R, S)- Proton δ (S, R, S) δ (S, R, R) δ (S, R, R) Ha 5.040 5.0290.011 Hb 2.486 2.511 −0.025 Hc, He Overlapping non- Overlapping non- NDequiv H's equiv H's Hd, Hd′ 0.912 0.876 0.036 He′ Overlap with BocOverlap with Boc ND Hf 3.209 3.212 −0.003 Hf′ 2.988 3.050 −0.062 HgOverlap with OMe Overlap with OMe ND Hg′ 3.364 3.389 −0.025 ND: notdeterminable Note that the first two letters correspond to the 2^(nd)eluting peak material and the third letter of the diastereomer refers tothe Mosher's ester chiral center.

SRS diastereomer: 1H, CDCl₃, 8 ppm 7.60-7.51 (m, 2H); 7.43-7.37 (m, 3H);5.04 (dd, J=8.0, 4.0, 1H); 3.52 (s, 3H); 3.51-3.45 (m, 1H); 3.36 (t,J=8.4, 1H); 3.28-3.12 (m, 1H); 3.07-2.90 (m, 1H); 2.59-2.39 (m, 1H);1.97-1.80 (m, 2H); 1.59-1.45 (m, 1H); 1.43 (s, 9H); 0.93 (d, J=6.8, 3H);0.90 (d, J=6.8, 3H).

SRR diastereomer: 1H, CDCl₃, δ ppm 7.62-7.52 (m, 2H); 7.44-7.36 (m, 3H);5.06-4.98 (m, 1H); 3.52 (s, 3H); 3.52-3.45 (m, 1H); 3.39 (t, J=8.8, 1H);3.30-3.14 (m, 1H); 3.10-2.96 (m, 1H); 2.60-2.40 (m, 1H); 1.96-1.80 (m,21-1); 1.58-1.45 (m, 1H); 1.43 (s, 9H); 0.96 (m, 6H).

Example 1 Crystalline Hydrochloride Salt of(S)-3-[(S)-1-(4-Chlorophenoxy)-2-methylpropyl]pyrrolidine

(S)-3-((S)-1-Hydroxy-2-methylpropyl)pyrrolidine-1-carboxylic acidt-butyl ester (2.6 g, 10.7 mmol, 1.0 eq.) and 1-chloro-4-fluorobenzene(3.4 mL, 32.0 mmol, 3.0 eq.) were dissolved in DMF (12 mL, 150 mmol).NaH (385 mg, 16.0 mmol, 1.5 eq.) was slowly added in three portions, andthe mixture was stirred at room temperature for 10 minutes undernitrogen. The mixture was heated at 90° C. for 3 hours, then cooled toroom temperature. The mixture was extracted with hexanes (50 mL) andwashed with water (50 mL). The aqueous layer was reextracted withhexanes (50 mL). The organic layers were combined, dried under Na₂SO₄,filtered, and concentrated. The crude BOC-protected intermediate wasthen purified by column chromatography (eluting with hexanes and ether,0-100%, flash chromatography). Deprotection was carried out using 1.2 MHCl in EtOH (150 mL, 180 mmol). The mixture was stirred at roomtemperature for 48 hours. The solution was concentrated until dry toyield the crude product as a mono-HCl salt. The crude mono-HCl salt wasdissolved in isopropanol (5 mL) to produce an oil, which was heated to55° C. Diisopropyl ether (25 mL) was slowly added under constantstirring to form a homogeneous solution, which was cooled to roomtemperature. The reaction vessel was scarred and seed crystals (fromheating and slowly cooling 100 mg of crude HCl salt using similarconditions) were added during the cooling process. Solids formed and thesolution was allowed to sit at room temperature for 1 hour. The solidswere filtered and washed with diisopropyl ether (10 mL) to yield a whitesolid (1.4 g). The filtrate was concentrated and crystallization wasrepeated twice to yield a total of 2.4 g (from 3 precipitations). Theprecipitate was dissolved in water and lyophilized to give the titlecompound as an off-white crystalline solid (2.4 g, 99% purity).

Alternate Preparation of Title Crystalline Hydrochloride Salt

(S)-3-((R)-1-hydroxy-2-methylpropyl)pyrrolidine-1-carboxylic acidt-butyl ester (35.0 g, 144 mmol, 1.0 eq.), triphenylphosphine (41.5 g,158 mmol, 1.1 eq.), p-chlorophenol (37.0 g, 288 mmol, 2.0 eq.) and2-methyl-tetrahydrofuran (300 mL) were combined and the vessel purgedwith nitrogen. Diisopropyl azodicarboxylate (31.2 mL, 1.1 eq.) was addedslowly at room temperature over 2 hours. The mixture was then stirred atroom temperature overnight. Hexanes (600 mL) were added and theresulting mixture was stirred at room temperature for 30 minutes. Thephases were separated and the organic layer was washed with 1.0 M NaOHin water (600 mL), washed with diluted saturated aqueous NaCl (20 mL),then dried over Na₂SO₄ to yield the crude BOC-protected intermediate.Additional hexanes (50 mL) were added and the resulting mixture wasstirred for 30 minutes. Solids were filtered off to yield the crudeBOC-protected intermediate as a thick oil, which was then purified bysilica gel chromatography (0-10-20% EtOAc in hexanes) to yield(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine-1-carboxylicacid t-butyl ester (5.5 g).

(S)-3-[(S)-1-(4-Chlorophenoxy)-2-methylpropyl]pyrrolidine-1-carboxylicacid t-butyl ester (24.7 g, 69.8 mmol, 1.0 eq.) was combined with 3 MHCl in cyclopentyl methyl ether (200 mL, 8.0 eq.). The mixture wasstirred at room temperature overnight. Most of the solvent was removedby rotary evaporation to yield a thick oil. Diisopropyl ether (300 mL)was added and the volume was then slowly reduced to ˜200 mL by rotaryevaporation to yield a slurry. The slurry was stirred at roomtemperature for 2 hours, then filtered and the filter cake was washedwith diisopropyl ether (50 mL) and dried to yield the title compound(18.1 g, 98.5% purity). The product was reslurried in 5 volumes of EtOAc(room temperature, heating to 50° C., then cooled to room temperature)to yield the title compound as a crystalline solid (18.0 g, >99%purity).

Example 2 Powder X-Ray Diffraction

A powder X-ray diffraction pattern (PXRD) was obtained with a RigakuMiniflex PXRD diffractometer using Cu Kα (30.0 kV, 15.0 mA) radiation.The analysis was performed with the goniometer running incontinuous-scan mode of 2° (20) per minute with a step size of 0.03°over a range of 2 to 40° in two-theta angle. Samples were prepared onquartz specimen holders as a thin layer of powdered material. Theinstrument was calibrated with a silicon metal standard, within ±0.20°two-theta angle. A representative PXRD pattern for the crystallinehydrochloride salt of the invention is shown in FIG. 1. The samples werehand ground prior to testing, in order to reduce particle sizeinterferences to the relative intensities.

The numerous intense powder diffraction peaks and relatively flatbaseline depicted in FIG. 1 strongly indicated that the crystallinehydrochloride salt possessed good crystallinity.

Example 3 Thermal Analysis

Differential scanning calorimetry (DSC) was performed using a TAInstruments Model Q-100 module with a Thermal Analyst controller. Datawere collected and analyzed using TA Instruments Thermal Solutionssoftware. A 1.22 mg sample of the crystalline hydrochloride salt of theinvention was accurately weighed into a covered aluminum pan. After a 5minute isothermal equilibration period at 22° C., the sample was heatedusing a linear heating ramp of 10° C./min from 22° C. to 250° C. Arepresentative DSC thermograph is shown in FIG. 2.

The DSC thermograph demonstrates that the crystalline hydrochloride salthas excellent thermal stability with a melting point at about 128° C.and no significant thermal decomposition below about 200° C.

A representative TGA trace is shown in FIG. 3, and indicates that asample of the crystalline hydrochloride salt did not lose anysignificant amount of weight from room temperature to 200° C., which isconsistent with the loss of residual moisture or solvent.

Example 4 Dynamic Moisture Sorption Assessment

A dynamic moisture sorption (DMS) assessment (also known as a moisturesorption-desorption profile) was performed for the crystallinehydrochloride salt of the invention using a VTI atmosphericmicrobalance, SGA-100 system (VTI Corp., Hialeah, FL 33016). A samplesize of approximately 2.73 mg was used and the humidity was set at theambient value at the start of the analysis. The DMS analysis consistedof a scan rate of 5% relative humidity/step over the full humidity rangeof 2% relative humidity to 90% relative humidity. The DMS run wasperformed isothermally at 25° C. A representative DMS profile is shownin FIG. 4.

The DMS profile demonstrates that the crystalline hydrochloride salt hasa reversible sorption/desorption profile with insignificanthygroscopicity. The crystalline hydrochloride salt has a small weightgain when it exposed to a broad humidity range from 2% relative humidityup to 90% relative humidity, and less than about 1.0% weight gain whenexposed to up to 85% relative humidity, which indicates that thecrystalline hydrochloride salt possesses only a minimal risk ofhygroscopicity below 85% relative humidity.

Example 5 Solubility and Stability

The crystalline hydrochloride salt of the invention has very goodaqueous solubility in a wide pH range:

SOLUBILITY CONCENTRATION (mg/mL) HCl pH 2.0 >10 50 mM phosphate-bufferedpH 7.4 >10 un-buffered water >10The crystalline hydrochloride salt of the invention (10 μg/mLconcentration) also has good stability with no loss of purity afterstorage for 3 days at 40° C.::

Solution Change in HPLC Peak Area (%) 50 mM citric acid pH 2.2 100.5 50mM phosphate-buffered pH 7.4 100.0The crystalline hydrochloride salt of the invention also has goodstability at room temperature with no degradation over 30 days atconcentrations of 1 mg/mL and 10 mg/mL.

Assay 1 hSERT, hNET, and hDAT Binding Assays

Membrane radioligand binding assays were used to measure inhibition oflabeled ligand (³H-citalopram or ³H-nisoxetine or ³H-WIN35428) bindingto membranes prepared from cells expressing the respective humanrecombinant transporter (hSERT or hNET or hDAT) in order to determinethe pK_(i) values of test compounds at the transporters.

Membrane Preparation from Cells Expressing hSERT, hNET, or hDAT

Recombinant human embryonic kidney (HEK-293) derived cell lines stablytransfected with hSERT or hNET, respectively, were grown in Dulbecco'sModified Eagle's Medium (DMEM) medium supplemented with 10% dialyzed FBS(for hSERT) or FBS (for hNET), 100 μg/ml penicillin, 100 μg/mlstreptomycin, 2 mM L-glutamine and 250 μg/ml of the aminoglycosideantibiotic G418, in a 5% CO₂ humidified incubator at 37° C. Whencultures reached 80% confluence, the cells were washed thoroughly in PBS(without Ca²⁺ and Mg²⁺) and lifted with 5 mM EDTA in PBS. Cells werepelleted by centrifugation, resuspended in lysis buffer (10 mM Tris-HCl,pH7.5 containing 1 mM EDTA), homogenized, pelleted by centrifugation,then resuspended in 50 mM Tris-HCl, pH 7.5 and 10% sucrose at 4° C.Protein concentration of the membrane suspension was determined using aBio-Rad Bradford Protein Assay kit. Membranes were snap frozen andstored at −80° C. Chinese hamster ovary membranes expressing hDAT(CHO-DAT) were purchased from PerkinElmer and stored at −80° C.

Binding Assays

Binding assays were performed in a 96-well assay plate in a total volumeof 200 μl assay buffer (50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.4)with 0.5, 1, and 3 μg membrane protein, for SERT, NET and DAT,respectively. Saturation binding studies, to determine radioligand K_(d)values for ³H-citalopram, ³H-nisoxetine, or ³H-WIN35428, respectivelywere conducted using 12 different radioligand concentrations rangingfrom 0.005-10 nM (³H-citalopram); 0.01-20 nM (³H-nisoxetine) and 0.2-50nM (³H-WIN35428). Inhibition assays for determination of pK_(a) valuesof test compounds were conducted with 1.0 nM ³H-citalopram, 1.0 nM³H-nisoxetine or 3.0 nM ³H-WIN35428, at 11 different concentrations oftest compound ranging from 10 μM to 100 μM.

Stock solutions (10 mM in DMSO) of test compound were prepared andserial dilutions made using Dilution Buffer (50 mM Tris-HCl, 120 mMNaCl, 5 mM KCl, pH 7.4, 0.1% BSA, 400 μM ascorbic acid). Non-specificradioligand binding was determined in the presence of 1 μM duloxetine, 1μM desipramine or 10 μM GBR12909 (each in Dilution Buffer) for thehSERT, hNET or hDAT assays, respectively.

Following a 60 minute incubation at 22° C. (or a period sufficient toreach equilibrium), the membranes were harvested by rapid filtrationover a 96-well UniFilter GF/B plate, pretreated with 0.3%polyethyleneimine, and washed 6 times with 300 μl wash buffer (50 mMTris-HCl, 0.9% NaCl, pH 7.5 at 4° C.). Plates were dried overnight atroom temperature, approximately 45 μl of MicroScint™-20 (Perkin Elmer)added and bound radioactivity quantitated via liquid scintillationspectroscopy. Inhibition curves and saturation isotherms were analyzedusing GraphPad Prism Software package (GraphPad Software, Inc., SanDiego, Calif.). IC₅₀ values were generated from concentration responsecurves using the Sigmoidal Dose Response (variable slope) algorithm inPrism GraphPad. K_(d) and B_(max) values for the radioligand weregenerated from saturation isotherms using the Saturation Binding GlobalFit algorithm in Prism GraphPad. pK_(i) (negative decadic logarithm ofK_(i)) values for test compounds were calculated from the best-fit IC₅₀values, and the IQ value of the radioligand, using the Cheng-Prusoffequation (Cheng & Prusoff (1973) Biochem. Pharmacol. 22(23):3099-3108):K_(i)=IC₅₀/(1+[L]/K_(d)), where [L]=concentration radioligand.

The compound of Example 1 (TFA salt) was tested in this assay and foundto exhibit a SERT pK_(a)≧8.0 and a NET pK_(i)≧8.0.

Assay 2 hSERT, hNET and hDAT Neurotransmitter Uptake Assays

Neurotransmitter uptake assays were used to measure inhibition of³H-serotonin (³H-5-HT), ³H-norepinephrine (³H-NE), and ³H-dopamine(³H-DA) uptake into cells expressing the respective transporter (hSERT,hNET or hDAT) in order to determine the pIC₅₀ values of test compoundsat the transporters.

³H-5-HT, ³H-NE, and ³H-DA Uptake Assays

HEK-293 derived cell lines stably-transfected with hSERT, hNET, or hDAT,respectively, were grown in DMEM medium supplemented with 10% dialyzedFBS (for hSERT) or FBS (for hNET and hDAT), 100 μg/ml penicillin, 100μg/ml streptomycin, 2 mM L-glutamine and 250 μg/ml of the aminoglycosideantibiotic G418 (for hSERT and hNET) or 800 μg/ml (for hDAT), in a 5%CO₂ humidified incubator at 37° C. When cultures reached 80% confluence,the cells were washed thoroughly in PBS (without Ca²⁺ and Mg²⁺) andlifted with 5 mM EDTA in PBS. Cells were harvested by centrifugation at1100 rpm for 5 minutes, washed once by resuspension in PBS, thencentrifuged. The supernatant was discarded and the cell pelletresuspended, by gentle trituration, in room temperature Krebs-Ringerbicarbonate buffer containing HEPES (10 mM), CaCl₂ (2.2 mM), ascorbicacid (200 μM) and pargyline (200 μM), pH 7.4. The final concentration ofcells in the cell suspension was 7.5×10⁴ cells/ml, 1.25×10⁵ cells/ml,and 5.0×10⁴ cells/ml for SERT, NET, and DAT cell lines, respectively.

Neurotransmitter uptake assays were performed in a 96-well assay platein a total volume of 400 μl assay buffer (Krebs-Ringer bicarbonatebuffer containing HEPES (10 mM), CaCl₂ (2.2 mM), ascorbic acid (200 μM)and pargyline (200 μM), pH 7.4) with 1.5×10⁴ and 2.5×10⁴ cells, forSERT, NET, and DAT, respectively. Inhibition assays for determination ofpIC₅₀ values of test compounds were conducted with 11 differentconcentrations, ranging from 10 μM to 100 μM. Stock solutions (10 mM inDMSO) of test compound were prepared and serial dilutions prepared using50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.4, 0.1% BSA, 400 μM ascorbicacid. Test compounds were incubated for 30 minutes at 37° C. with therespective cells, prior to addition of radiolabeled neurotransmitter,³H-5-HT (20 nM final concentration), ³H-NE (50 nM final concentration)or ³H-DA (100 nM final concentration). Non-specific neurotransmitteruptake was determined in the presence of 2.5 μM duloxetine, 2.5 μMdesipramine, or 10 uM GBR-12909 (each in Dilution Buffer) for the hSERT,hNET, or hDAT assays, respectively.

Following a 10 minute incubation, at 37° C., with radioligand, the cellswere harvested by rapid filtration over a 96-well UniFilter GF/B plate,pretreated with 1% BSA, and washed 6 times with 650 μl wash buffer (icecold PBS). Plates were dried overnight at 37° C., ˜45 μl ofMicroScint-20 (Perkin Elmer) added and incorporated radioactivityquantitated via liquid scintillation spectroscopy. Inhibition curveswere analyzed using GraphPad Prism Software package (GraphPad Software,Inc., San Diego, Calif.). IC₅₀ values were generated from concentrationresponse curves using the Sigmoidal Dose Response (variable slope)algorithm in Prism GraphPad.

The compound of Example 1 (TFA salt) was tested in this assay and foundto exhibit a serotonin reuptake inhibition pIC₅₀ value of ≧8.0 and anorepinephrine reuptake inhibition pIC₅₀ value of ≧8.0.

Assay 3 Ex Vivo SERT and NET Transporter Occupancy Studies

Ex vivo radioligand binding and neurotransmitter uptake assays are usedto determine the in vivo occupancy of SERT and NET, in selected brainregions, following in vivo administration (acute or chronic) of testcompounds. Following administration of test compound (by intravenous,intraperitoneal, oral, subcutaneous or other route) at the appropriatedose (0.0001 to 100 mg/kg), rats (≧n=4 per group) are euthanized atspecific time points (10 minutes to 48 hours) by decapitation and thebrain dissected on ice. Relevant brain regions are dissected, frozen andstored at −80° C. until use.

Ex Vivo SERT and NET Radioligand Binding Assays

For ex vivo radioligand binding assays, the initial rates of associationof SERT (³H-citalopram), and NET (³H-nisoxetine) selective radioligandswith rat brain crude homogenates, prepared from vehicle and testcompound-treated animals, are monitored. See Hess et al. (2004) J.Pharmacol. Exp. Ther. 310(2):488-497. Crude brain tissue homogenates areprepared by homogenizing frozen tissue pieces in 0.15 ml (per mg wetweight) of 50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.4 buffer.Radioligand association assays are performed in a 96-well assay plate ina total volume of 200 μA assay buffer (50 mM Tris-HCl, 120 mM NaCl, 5 mMKCl, 0.025% BSA, pH 7.4) with 650 μg wet weight tissue (equivalent to 25μg protein). Homogenates are incubated for up to 5 minutes with³H-citalopram (3 nM) and ³H-nisoxetine (5 nM), respectively, prior totermination of the assay by rapid filtration over a 96-well UniFilterGF/B plate, pretreated with 0.3% polyethyleneimine. Filters then arewashed 6 times with 300 μl wash buffer (50 mM Tris-HCl, 0.9% NaCl, pH7.4 at 4° C.). Non-specific radioligand binding is determined in thepresence of 1 μM duloxetine, or 1 μM despiramine, for ³H-citalopram or³H-nisoxetine, respectively. The plates are dried overnight at roomtemperature, ˜45 μl of MicroScint™-20 (Perkin Elmer) is added and boundradioactivity quantitated via liquid scintillation spectroscopy. Theinitial rates of association of ³H-citalopram and ³H-nisoxetine aredetermined by linear regression using GraphPad Prism Software package(GraphPad Software, Inc., San Diego, Calif.). The average rate ofradioligand association to brain tissue homogenates from vehicle-treatedanimals us determined. The % occupancy of test compounds then usdetermined using the following equation:

% occupancy =100×(1−(initial rate association for test compound-treatedtissue/mean rate association for vehicle-treated tissue))

ED₅₀ values are determined by plotting the log 10 of the dose of thetest compound against the % occupancy. ED₅₀ values are generated fromconcentration response curves using the Sigmoidal Dose Response(variable slope) algorithm in GraphPad Prism.

Ex Vivo SERT and NET Uptake Assays

Ex vivo neurotransmitter uptake assays, in which the uptake of ³H-5-HTor ³H-NE into rat brain crude homogenates, prepared from vehicle andtest compound-treated animals, are used to measure in vivo SERT and NETtransporter occupancy. See Wong et al. (1993) Neuropsychopharmacology8(1):23-33. Crude brain tissue homogenates are prepared by homogenizingfrozen tissue pieces in 0.5 ml (per mg wet weight) of 10 mM HEPES bufferpH 7.4, containing 0.32 M sucrose, 200 μM ascorbic acid and 200 μMpargyline, at 22° C. Neurotransmitter uptake assays are performed in a96-well Axygen plate in a total volume of 350 μl assay buffer(Krebs-Ringer bicarbonate buffer with 10 mM HEPES, 2.2 mM CaCl₂, 200 μMascorbic acid and 200 μM pargyline, pH 7.4) with 50 μg protein.Homogenates are incubated for 5 minutes at 37° C. with ³H-5-HT (20 nM)and ³H-NE (50 nM), respectively, prior to termination of the assay byrapid filtration over a 96-well UniFilter GF/B plate, pretreated with 1%BSA. Plates are washed 6 times with 650 μl wash buffer (ice cold PBS)and dried overnight at 37° C., prior to addition of 45 μl ofMicroScint™-20 (Perkin Elmer). Incorporated radioactivity is quantitatedvia liquid scintillation spectroscopy. Non-specific neurotransmitteruptake is determined in parallel assays in which tissue homogenates areincubated with ³H-5-HT (20 nM) or ³H-NE (50 nM) for 5 minutes at 4° C.

Assay 4 Other Assays

Other assays that are used to evaluate the pharmacological properties oftest compounds include, but are not limited to, cold ligand bindingkinetics assays (Motulsky and Mahan (1984) Molecular Pharmacol.25(1):1-9) with membranes prepared from cells expressing hSERT or hNET;conventional membrane radioligand binding assays using radiolabeled, forexample, tritiated, test compound; radioligand binding assays usingnative tissue from, for example rodent or human brain; neurotransmitteruptake assays using human or rodent platelets; neurotransmitter uptakeassays using crude, or pure, synaptosome preparations from rodent brain.

Assay 5 Formalin Paw Test

Compounds are assessed for their ability to inhibit the behavioralresponse evoked by a 50 μl injection of formalin (5%). A metal band isaffixed to the left hind paw of male Sprague-Dawley rats (200-250 g) andeach rat is conditioned to the band for 60 minutes within a plasticcylinder (15 cm diameter). Compounds are prepared in pharmaceuticallyacceptable vehicles and administered systemically (i.p., p.o.) atpre-designated times before formalin challenge. Spontaneous nociceptivebehaviors consisting of flinching of the injected (banded) hind paw arecounted continuously for 60 minutes using an automated nociceptionanalyzer (UCSD Anesthesiology Research, San Diego, Calif.).Antinociceptive properties of test articles are determined by comparingthe number of flinches in the vehicle and compound-treated rats (Yaksh TL et al., “An automated flinch detecting system for use in the formalinnociceptive bioassay” (2001) J. Appl. Physiol. 90(6):2386-2402).

Assay 6 Spinal Nerve Ligation Model

Compounds are assessed for their ability to reverse tactile allodynia(increased sensitivity to an innocuous mechanical stimulus) induced bynerve injury. Male Sprague-Dawley rats are surgically prepared asdescribed in Kim and Chung “An experimental model for peripheralneuropathy produced by segmental spinal nerve ligation in the rat”(1992) Pain 50(3):355-363. Mechanical sensitivity is determined as the50% withdrawal response to innocuous mechanical stimuli (Chaplan et al.,“Quantitative assessment of tactile allodynia in the rat paw” (1994) J.Neurosci. Methods 53(1):55-63) before and after nerve injury. One tofour weeks post-surgery, compounds are prepared in pharmaceuticallyacceptable vehicles and administered systemically (i.p., p.o.). Thedegree of nerve injury-induced mechanical sensitivity before and aftertreatment serves as an index of the compounds' antinociceptiveproperties.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

1. (canceled)
 2. The method of claim 14, where the crystalline salt ischaracterized by having one or more additional diffraction peaks at 2θvalues selected from 26.42±0.20, 30.65±0.20, 28.91±0.20, 24.77±0.20,14.42±0.20, 16.74±0.20, and 5.20±0.20.
 3. The method of claim 14, wherethe crystalline salt is characterized by a powder x-ray diffractionpattern in which the peak positions are substantially in accordance withthe peak positions of the pattern shown in FIG.
 1. 4. The method ofclaim 14, where the crystalline salt is characterized by a differentialscanning calorimetry trace which has a melting point of about 128° C. 5.The method of claim 14, where the crystalline salt is characterized by adifferential scanning calorimetry trace substantially in accordance withthat shown in FIG.
 2. 6-13. (canceled)
 14. A method of treating apatient that is suffering from attention deficit hyperactivity disordercomprising administering a therapeutically effective amount of acrystalline salt of(S)-3-[(S)-1-(4-chlorophenoxy)-2-methylpropyl]pyrrolidine andhydrochloric acid in a 1:1 molar ratio, characterized by a powder x-raydiffraction pattern comprising diffraction peaks at 2θ values of8.78±0.20, 15.26±0.20, 19.08±0.20, 20.36±0.20, 21.50±0.20, and25.46±0.20.
 15. (canceled)